Hi, Guys, I noticed that researchers usually permeabilized the cells with 0.1% Triton-X100 or ice-cold methanol (90%) for an intracellular protein staining, it looks like that the two methods work very well and there is not anyone to specify the difference.
Now I'm wondering what's the difference between 0.1% triton-X100 and ice-cold methanol in the immunofluorescence assay for intracellular protein staining?
Hi Fusheng si
Methanol and other organic solvents dissolve lipids from the cell membranes making the permeable for antibody staining. They can also be used to fix cells simultaneously. However, methanol is not good for staining soluble proteins. Therefore, researchers can use detergents instead.
Detergents include Triton-X100 and Tween-20 and tend to displace lipids altogether which can also include proteins. If your protein is membrane-bound, this can result in loss of signal. Tween-20 is more gentle than Triton-X100 and has less loss of signal to membrane-bound proteins. However, it is not strong enough to permeabilise the nucleus - thus Triton is used here.
More information is found here: https://link.springer.com/protocol/10.1007%2F978-1-59745-324-0_9
Hope this helps!
Hi Fusheng si
Methanol and other organic solvents dissolve lipids from the cell membranes making the permeable for antibody staining. They can also be used to fix cells simultaneously. However, methanol is not good for staining soluble proteins. Therefore, researchers can use detergents instead.
Detergents include Triton-X100 and Tween-20 and tend to displace lipids altogether which can also include proteins. If your protein is membrane-bound, this can result in loss of signal. Tween-20 is more gentle than Triton-X100 and has less loss of signal to membrane-bound proteins. However, it is not strong enough to permeabilise the nucleus - thus Triton is used here.
More information is found here: https://link.springer.com/protocol/10.1007%2F978-1-59745-324-0_9
Hope this helps!
Hi Fusheng
I agree to what Jack has said. I use both methods depending on what i want to see. 0.1% Triton X-100 is usually standard and works well for visualization of most intracelular proteins. Those proteins which are bound by several membrane however, do not stain well when permeabilized with 0.1% Triton X-100.
For such cases, fixation with ice-cold methanol at -20C for 5 min works wonderfully. Methanol is massively dehydrating, so it shrinks the nucleus and organelles. It also strips out lipids and denatures protein, thereby improving epitope accessibility for several targets in the cell. Since methanol does both fixation and permeabilization, there is no separate step for permeabilization. However, do ensure to quench excess aldehyde using 0.1% Glycine in PBS, or it may interfere with antibodies.
Hope this helps
Best
Aparajita
Under normal conditions, antibody molecules are too large and ionic to pass through the cellular membrane to detect intracellular proteins. Permeabilizing the cells through methanol or acetone fixation, or with the use of a detergent, allows antibodies to pass through the cellular membrane and enter the cell. The most common reagent used for cell permeabilization is non-ionic detergent, Triton X-100. Other milder permeabilizing agents include digitonin or related saponin compounds.
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